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Figure 5 | BMC Biochemistry

Figure 5

From: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5

Figure 5

Polyubiquitylation of Myf5 is required for its degradation. (A) Myf5 is polyubiquitylated in mitotic extracts: Myf5 was incubated in a mitotic extract in the presence of ubiquitin (1 mg/ml), MG132 (200 μM) and ubiquitin aldehyde (5 μM). At the times indicated, 2 μl samples were analyzed by 10% SDS-PAGE to visualize the formation of high-MW adducts ((Ub)n-Myf5, poly-ubiquitylated Myf5 molecules). (B) Lysine-less (K0) ubiquitin inhibits Myf5 polyubiquitylation: Myf5 ubiquitylation was performed in a mitotic extract in the presence of MG132 (200 μM), ubiquitin aldehyde (1 μM), ubiquitin (Ub) and lysine-less-ubiquitin (UbK0) at the indicated concentrations, for 30 min at 25°C. (C) Lysine-less ubiquitin stabilizes Myf5: Degradation assays were performed in 20 μl reaction mixtures containing 16 μl of mitotic extract, 2 μl of radiolabeled in vitro translated Myf5 and a mixture of wild type and K0 ubiquitin as indicated. The reaction was performed at 25°C for 40 min, 2 μl samples were resolved by 10% SDS-PAGE. "START" corresponds to a 2 μl sample taken at time 0 from the reaction containing 1 mg/ml Ub. Radioactivity was quantified as previously described (see legend of figure 2) and percentages of degradation were calculated for each Ub/UbK0 ratio from the difference between the times 0 (not shown) and 40 minutes.

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